In various areas of medical diagnostics there is an urgent need for new technology capable of reducing time and cost of existing analytical tools. For example, standard diagnostic tests for infectious disease are not sufficiently rapid for early diagnosis of sepsis, a life-threatening systemic disease affecting each year approximately 400,000 individuals in the U.S. alone. In most patients, septic shock occurs when gram-negative bacteria enter the blood stream following local bacterial infections such as meningitis, pneumonia, and urinary tract infections. Clinical data indicate that early diagnosis of sepsis is crucial because the risk of death increases substantially when treatment is delayed.
Standard bacteriological tests need 24-48 hours for completion because they require a preliminary amplification/purification step in which a specimen is first cultured in agar until visible bacterial colonies appear. Subsequently, one or more of the bacterial colonies is collected and tested for antibiotic resistance and/or bacterial identification markers.
Progress in the last two decades in this field has been mainly limited to improving the process of colony testing using automated analyzers based on chromogenic and fluorogenic enzymatic reactions. The currently available analyzers, however, still require about one fourth of a bacterial colony (about 2×107 cells) for each biochemical test, and bacterial identification takes 3-12 hours.
The BCR-methodology disclosed in U.S. Pat. No. 5,472,846 and the amplification system of the present invention are radically different methodologies although both make use of living microorganisms for amplification. In contrast to BCR: (1) the present invention does not require growth of vegetative bacterial cells since it depends exclusively on spore germination; (2) this invention does not require enzyme-labeled probes; (3) the chain reaction in this invention consists of a propagating cascade of spore germination generated through de novo synthesized or activated enzymes acting on a germinogenic source present in the reaction mixture whereas the chain reaction in BCR consists of bacterial proliferation generated through enzymatic destruction of a growth inhibitor, typically an antibiotic, present in the reaction mixture; and (4) this invention requires spores and cannot operate with vegetative cells, while BCR can operate equally well with spores or vegetative cells.
Another methodology which makes use of bacterial spores is disclosed by N. Citri in U.S. Pat. No. 5,614,375. Citri teaches detection of biotoxic contaminants based upon their inhibitory effect on enzyme synthesis which occurs de novo during spore germination. The differences between Citri and the present invention become clear when testing bacteria or other particulate analytes since Citri's methodology is not capable of either detecting or identifying these types of analytes whereas the present invention does so.
Accordingly, it is an object of the present invention to provide an improved biological/biochemical assay for determining the presence of various microorganisms, viruses, nucleic acids, and polypeptides in a test sample.